Mammalians have two major types of sensory neurons in the retina: rods, specialized for vision in dim-light, and cones for vision in well-lit conditions and the perception of color. Most mammals have two cone types, namely S- and M-cones. They diverge in their sensitivity to different wavelengths of light, based on their expression of different light-sensitive proteins: S-opsin for blue light and M-opsin for green light. The purpose of this project is to identify genetic differences in cones and, more specifically, to find molecules that are involved in cone synapse formation. Until now, cones have been classified mostly by the opsin they express. Recent findings in our laboratory point to the existence of additional genetic differences. Normally, the dendrites of the S-cone bipolar cell (SCBC), an interneuron that relays cone-signals to downstream neurons, exclusively contact S-cones. However, genetic disruption of the normal S- and M-opsin expression pattern in different knockout mice fails to alter that specific connection. Can we identify genes other than the opsins that are uniquely expressed in each cone type? And, can we identify molecules that facilitate the formation of the specific S-cone/SCBC synapse? To identify such molecular signatures, we have turned to single cell RNA-seq to obtain complete genetic profiles of S- and M-cones. For this study, we used the 13-lined ground squirrel that, in contrast to mouse, is diurnal and has a cone-dominated retina. The two cone types are morphologically indistinguishable, so we developed a protocol to dissociate and label live cells with an antibody targeting the extracellular domain of S-cones We then manually collected single cells for next-generation sequencing. The analysis reveals differentially expressed genes that define cone identity beyond their expression of S- or M-opsins. We are validating these candidate genes for further investigation. In collaborate with Drs. Lijin Dong and Grime Wistows group at NEI, we also studied the role of miRNA-183/96/182 cluster in the development of cone photoreceptors and identified a severe cone maturation defect in the KO mice that affects both S- and M-cones. Further expansion of the work from retina to other sensory system unveiled a general role of this miRNA cluster in sensory receptor development of multiple systems. We plan to continue the investigation using the above-mentioned cone single cell sequencing technique.